Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective therapeutics. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrP-res or PrPSc) of the normal, protease-sensitive prion protein, PrP-sen. PrP-res can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of prion disease appear to be less transmissible than non-amyloid forms, suggesting that there is a fundamental difference in how they trigger disease. Furthermore, it is unclear whether or not prion diseases where PrP-res is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrP-res is deposited as non-amyloid forms. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses on: 1) understanding the pathways of PrP amyloid formation and, 2) studying how mutations in PrP influence PrP-res amyloid formation in familial forms of prion disease. In 2011, using LC-MS/MS Nanospray Ion Trap Mass Spectrometry we generated the proteomes of prion infected mouse brain tissue which had accumulated PrP-res in either amyloid or non-amyloid forms. We also completed a series of long term (i.e. >2 years) in vivo experiments where transgenic mice expressing human PrP-sen with or without the GPI anchor were infected with different types of human prion and other amyloid diseases. We are currently processing and analyzing tissues from these experiments using standard biochemical, histopathological and immunohistochemical techniques. Analysis of data from the proteomic and in vivo studies is ongoing and should provide insight into whether or not amyloid and non-amyloid forms of prion disease differ mechanistically. Hereditary forms of prion disease are associated with mutations within the PrP gene. One of these mutations is the insertion of extra copies of an eight amino acid motif (octapeptide repeat) into PrP. We have used an in vitro fibrillization model of this hereditary mutation to study how the repeat region influences the formation of both PrP-res and PrP amyloid. Last year, we gathered data suggesting that the C-terminus could influence the structure of the octapeptide repeat region in PrP amyloid. In 2011, we used standard biochemical techniques and PrP peptides associated with human prion disease to show that the C-terminus of PrP appears to have this effect during the initial folding of PrP-sen and not during the fibrillization of PrP into amyloid. These data will help to define the steps involved in the refolding of normal PrP into amyloid. Furthermore, they may help to explain why the structure of the octapeptide repeat region appears to be strain specific and thus may help to explain the different pathologies associated with different types of prion infectivity. Finally, in 2011 we introduced into the laboratory an in vitro system that has been shown to generate infectious prions from recombinant PrP-sen (Science 327: 1132 (2010)). We will use this system to 1) study how protease resistant PrP is made and, 2) define the currently unknown fundamental nature of the infectious prion particle.